• Nuclear Medicine and Molecular Imaging
• /
• v.42 no.5
• /
• pp.394-400
• /
• 2008

Purpose: Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirus-mediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Materials and Methods: Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was $19.1{\pm}4.7%$, $54.0{\pm}6.4%$, $85.7{\pm}8.7%$, and $98.4{\pm}1.3%$ at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and 1-125 uptake. Results: Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro 1-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC ($29,704{\pm}6,659\; picomole/10^6\;cells$) was greater than that in adeno-hNIS-rMSC at MOI 100 ($6,168{\pm}2,134\;picomole/10^6\;cells$). Conclusion: Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression.

• Nuclear Medicine and Molecular Imaging
• /
• v.43 no.5
• /
• pp.468-477
• /
• 2009

Purpose: Hydrodynamic-based procedure is a simple and effective gene delivery method to lead a high gene expression in liver tissue. Non-invasive imaging reporter gene system has been used widely with herpes simplex virus type 1 thymidine kinase (HSV1-tk) and its various substrates. In the present study, we investigated to image the expression of HSV1-tk gene with 5-(2-iodovinyD-2'-deoxyuridine (IVDU) in mouse liver by the hydrodynamicbased procedure. Materials and Methods: HSV1-tk or enhanced green fluorescence protein (EGFP) encoded plasmid DNA was transferred into the mouse liver by hydrodynaminc injection. At 24 h post-injection, RT-PCR, biodistribution, fluorescence imaging, nuclear imaging and digital wholebody autoradiography (DWBA) were performed to confirm transferred gene expression. Results: In RT-PCR assay using mRNA from the mouse liver, specific bands of HSV1-tk and EGFP gene were observed in HSV1-tk and EGFP expressing plasmid injected mouse, respectively. Higher uptake of radiolabeled IVDU was exhibited in liver of HSV1-tk gene transferred mouse by biodistribution study. In fluorescence imaging, the liver showed specific fluorescence signal in EGFP gene transferred mouse. Gamma-camera image and DWBA results showed that radiolabeled IVDU was accumulated in the liver of HSV1-tk gene transferred mouse. Conclusion: In this study, hydrodynamic-based procedure was effective in liver-specific gene delivery and it could be quantified with molecular imaging methods. Therefore, co-expression of HSV1-tk reporter gene and target gene by hydrodynamic-based procedure is expected to be a useful method for the evaluation of the target gene expression level with radiolabeled IVDU.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.31 no.3
• /
• pp.193-197
• /
• 2009

Background: Though it is clear that many types of viruses can infect the oral mucosa, its condition for infection is unclear. The purpose of this study was to analyze the conditions for viral infection of normal oral mucosa and explore the possibility of topical gene therapy to oral mucosa using a viral vector. Methods: Freshly taken fragments of the palate and the tongue of mice were used for organ culture. The specimens were exposed to green fluorescent protein (GFP)-adenoviral vector for 1 hour except for the control. Initial viral titer was $6.3{\times}10^{11}\;pfu/ml$ and the virus was diluted to working concentrations. The dilution ratio was 1:1,000 ($6.3{\times}10^8\;pfu/ml$), 1:10,000 ($6.3{\times}10^7\;pfu/ml$), and 1:100,000 ($6.3{\times}10^6\;pfu/ml$). They were then cultured on a stainless steel wire mesh in an organ culture dish. The specimens were stereoscopically examined every 24 hours for 6 days, after which they were fixed and analyzed through immunohistochemical methods Results: There was no visible expression in the control, $6.3{\times}10^6\;pfu/ml$, and $6.3{\times}10^7\;pfu/ml$ groups. Initial expression was observed at 24 hours after infection in both the palate and the tongue in $6.3{\times}10^8\;pfu/ml$ and the expression significantly increased until 3 days in the palate and 2 days in the tongue after infection (P<0.05). In both groups, the expression was mostly observed at the resection margin. Immunohistochemical studies showed that the epithelial cells were positive to GFP. Conclusion: The present study showed that topically applied adenovirus containing specific genetic information of GFP could successfully transduce GFP in normal oral epithelial cells at the resection margin in organ culture in terms of dose and exposure time.

• Asian-Australasian Journal of Animal Sciences
• /
• v.20 no.9
• /
• pp.1407-1416
• /
• 2007

Ten, first lactation, 87.5%HF dairy cattle were used to investigate effects of long-term administration of recombinant bovine somatotropin (rbST) on nutrient uptake by the mammary gland at different stages of lactation. Measurements of arterial plasma concentrations and arterial-venous differences of metabolites across the mammary gland were performed in combination with measurment of mammary blood flow to estimate the mammary uptake. Animals in experimental groups were injected subcutaneously every 14 days from day 60 of lactation with a prolonged-release formulation of 500 mg of rbST (POSILAC, Monsanto, USA) or with sterile sesame oil without rbST in the control group. During early lactation, the milk yield of rbST-treated animals was higher than that of the control animals (p<0.05). The peak milk yield in both groups of animals declined from the early period of lactation with progression to mid- and late-lactation. No significant changes were observed in the concentration of milk lactose, while the concentrations of milk protein significantly increased as lactation advanced to mid- and late-lactation in both groups. Milk fat concentrations were significantly higher in rbST-treated animals than in control animals, particularly in early lactation (p<0.05). Mammary blood flow (MBF) markedly increased during rbST administration and was maintained at a high level throughout lactation. The mean arterial plasma concentrations for glucose and acetate of rbST-treated animals were unchanged. The net mammary glucose uptake of rbST-treated animals increased approximately 20% during early lactation, while it significantly decreased (p<0.05), including the arteriovenous differences (A-V differences) and extraction ratio across the mammary gland, as lactation advanced to mid- and late-lactation. A-V differences, mammary extraction and mammary uptake for acetate increased during rbST administration and were significantly higher (p<0.05) than in the control animals in early and mid-lactation. Mean arterial plasma concentrations for ${\beta}$-hydroxybutyrate and free glycerol were unchanged throughout the experimental periods in both groups. A-V differences and extraction ratio of ${\beta}$-hydroxybutyrate across the mammary gland did not alter during rbST administration. Mean arterial plasma concentrations for free fatty acids ($C_{16}$ to $C_{18}$), but not for triacylglycerol, increased in rbST-treated animals and were significantly higher than in control animals during early lactation (p<0.01). These findings suggest that an increase in MBF during rbST administration would not be a major determinant in the mediation of nutrient delivery and uptake by the mammary gland for increased milk production. Local changes in biosynthetic capacity within the mammary gland would be a factor in the utilization of substrates resulting in the rate of decline in milk yield with advancing lactation.

• Journal of Radiopharmaceuticals and Molecular Probes
• /
• v.5 no.2
• /
• pp.89-100
• /
• 2019

In our previous studies, we developed a ¹⁸F-labeled TSPO-binding ligand, named [¹⁸F]CB251, which has been proved to be a promising TSPO-binding PET radiotracer for the detection and monitoring of TSPO expression in pathological diseases. (Ki = 0.27 nM for TSPO, 1.96% ID/g of tumor uptake at 1h post-injection) Based on these results, we utilized 6,8-dichloro-2-phenylimidazo[1,2-a]pyridineacetamide analogs, CB185 (1) as a targeting moiety for the selective delivery of probes and anticancer molecules to TSPO-overexpressed tissues. In this study, we designed CB185 derivatives contains different PEG chains (n = 1, 3 and 5) and fluorescence dye (Cy5) to identify the necessary space between a TSPO-binding ligand and an anticancer agent. Three CB185 derivatives (11a-c) which contains Cy5 and PEG chain, were synthesized and the effect of PEG additive on their TSPO-binding affinities were evaluated using in vitro assays. The binding affinity for compounds 11a-c was lower than that of PK11195 (Ki = 3.2 nM), but still characterized by nanomolar binding affinity for TSPO (Ki = 46.5 nM for 11a, 51.0 nM for 11b, and 388.5 nM for 11c). These results showed that the conjugates are characterized by a moderate binding affinity toward TSPO except for compound 11c, which PEG chain consist of five PEG monomers. Our finding might add useful information to decide the appropriate PET chain length for developing new TSPO-targeting drug carriers.

• Korean Journal of Poultry Science
• /
• v.35 no.3
• /
• pp.241-245
• /
• 2008

Lentiviral vector system is efficient vehicles for the delivery of exogenous genes, and it is generally used in the generation of transgenic chickens. In this study, we used recombinant lentiviral vectors to generate transgenic chicks that express the human superoxide dismutase-3 gene driven by the chicken ovalbumin promoter. It is well known that superoxide dismutases(SODs) are believed to play a crucial role in protecting cells against oxygen toxicity. There are three forms of SOD proteins: cytosolic Cu-Zn SOD, mitochondrial Mn SOD, and extracellular SOD(SOD-3). The recombinant lentivirus containing the human SOD-3 gene was injected into the subgerminal cavity of freshly laid eggs. Subsequently, the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. From 341 injected embryos, the 78 chicks hatched after 21 days incubation. The hatched chicks were screened for the human SOD-3 gene by using PCR. Two of 47 male chickens that survived to sexual maturity contained the human SOD-3 gene in their semen. These results showed that our transgenic chicken generation system was completely established.

• Reproductive and Developmental Biology
• /
• v.30 no.2
• /
• pp.87-92
• /
• 2006

The possibility of producing transgenic embryos expressing the green fluorescence protein (GFP) gene have been evaluated after transfer of exogenous gene into the porcine zygote cytoplasm using the intracytoplasm sperm injection (ICSI) as gene delivery method. For DNA binding to sperm heads, 0.05% Triton X-100 or Lipofectin was used. After injection of the sperm bound to DNA by means of Lipofectin or Triton X-100 triturate, the blastocyst formation rates on day 6 were not significantly different from that of ICSI only group (18.8, 19.2 and 25.3%). In terms of GFP expression, more embryos were in GFP form in Triton X-100 group than in Lipofectin group (40.6 vs 36.4%), while percentage of non-mosaic embryos expressing the GFP gene in all blastomere was higher (P<0.05) in Lipofectin group than in Triton X-100 group (4.2 vs 0.9%). ICSI embryos derived from sperm treated with Lipofectin/DNA complex was transferred into 3 recipients and were collected by uterine flushing on days 5, 7 and 15 after embryo transfer, and then GFP expression was observed by a fluorescence microscopy. Over 26% of the collected embryos were normally expressed GFP gene. These results suggest that foreign gene transfer method with DNA bound sperm caused minimal damage to structure of oocytes that can result to full development of porcine embryos. This was confirmed in this study when the embryos that were transferred after ISCI of DNA bound sperm had a normal development and gene expression until preimplantation.

• Journal of Nutrition and Health
• /
• v.36 no.7
• /
• pp.736-742
• /
• 2003

Soybeans have been a major protein source for many centuries in Korea. Soybeans contain phytochemicals which are isoflavones, biochemically active component. Isoflavone is a kind of phytoestrogen, structurally and functionally similar to estrogen. It has been reported that the breast milk and blood of breast feeding mothers who consume soy products contain isoflavones. This study was conducted to investigate the effects of soy milk supplement on the isoflavones (daidzein, genistein) concentration of breast milk, plasma and urine from breast feeding woman. Seventeen healthy women who delivered at Kyung Hee Medical Center were recruited. For the first 2 weeks after delivery, seventeen women ingested 400 ml (isoflavone 43.2 mg) of soy milk on the given time starting from the day of giving birth. For the next 2 weeks, soy milk ingestion was withdrawn. Dietary intake and anthropometric data were checked and breast milk, blood, and 24 hr urine samples were collected on the day of giving birth, the 14th (the last day of the supplement phase) and 28th (the last day of the withdrawal phase) day, respectively. HPLC analysis was used to measure the concentration of isoflavones. Dietary intakes of the subjects were inadequate for the Korean RDA regardless of soy milk supplementation. Especially, intakes of vit A, calcium, and iron were very low. The Anthropometric data such as LBM, TBW, PIBW, BMI checked on the day of 14th decreased and maintained their levels by the 28th day. Daidzein concentration in breast milk was not affected by soy milk supplementation. However, genistein concentration decreased by the 28th day (14th day: 0.89 $\pm$ 0.10 $\mu$g/ml, 28th day : 0.48 $\pm$ 0.07 $\mu$g/ml) (p < 0.05). Plasma daidzein and genistein concentrations were not changed by the 14th day and decreased by the 28th day (14th day: 49.64 $\pm$ 3.30 ng/ml, 26.72 $\pm$ 2.90 ng/ml, 28th day: 38.30 $\pm$ 4.40 ng/ml, 6.51 $\pm$ 0.50 ng/ml, respectively) (p < 0.05). Twenty four hour urine concentrations of daidzein and genistein significantly increased by the 14th day and decreased by the 28th day (14th day: 5.80 :t 0.3 mg/d, 4.17 $\pm$ 0.2 mg/d, 28th day: 6.72 $\pm$ 0.4 mg/d, 5.09 $\pm$ 0.5 mg/d, respectively) (p < 0.001). The rate of urinary recovery of daidzein was greater than that of genistein. The results of this study indicate that the supplement of dietary soy milk to the lactating women elevates the contents of isoflavone in the breast milk.

• Journal of Yeungnam Medical Science
• /
• v.3 no.1
• /
• pp.41-48
• /
• 1986

Band 3, the predominent 95,000 dalton anion transport protein, is the major intrinsic glycoprotein of the human erythrocyte membrane. This anion carrier exists as a dimer and binds the cytoskeletons such as spectrin, ankyrin and actin. And the liposomes are vesicular structures which form spontaneouly upon hydration of phospholipids. These artificial lipid vesicles have been investigated as model of the biological membranes and as a mean of improving the delivery of nucleic acids, drugs, proteins and biological substances to specific target tissues and cells. In this study, we were purified Band 3 from the human erythrocyte membrane(ghost) was prepared by hemolysis of intact human erythrocyte with weak alkali-hypotonic solution. Band 6 was removed from ghost by extracting with solution of an ionic strength of 0.15. Band 3 and Band 4 were solubilized selectively by extracting Band 6-depleted ghosts with Triton X-100 under nondenaturing conditions. Band 3 was then purified from Triton X-100 extract treated with p-chloromercuribenzoate by sucrose density gradient ultracentrifugation. This purified Band 3 was incorporated into liposomes prepared by reverse-phase evaporation. Phosphatidyl L-serine and cholesterol(1 : 1 molar ratio) were dissolved in chloroform and then chloroform was removed by rotatory evaporation under reduced pressure. Band 3 solution without Triton X-100 was introduced into a mixture of lipids and diethylether. Diethylether was subsequently removed by evaporation. This purified Band 3 and its incorporation into liposomes were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

• Journal of the Korean Dietetic Association
• /
• v.6 no.2
• /
• pp.136-147
• /
• 2000

The aim of this research was to examine the management practices related to foodservice.clinical nutrition service provided by hospital foodservice operations. A survey of 30 hospital food nutrition service departments was undertaken and detailed information was collected from each, including surveys of 176 dietitians and 30 foodservice managers. Statistical data analysis was completed using the SAS/win 6.11 package for descriptive analysis and t-test. The results of this study can be summarized as follows : Out of 30 hospitals, 73.3%(22) were directly operated and 26.7%(8) were under contract foodservice management. Licensed number of beds were 768.7, with an average length of 11.4 days. The general characteristics of the dietitians were that 49.4% were aged between 25-29, and 60.8% were ordinary dietitians. 76.7% had bachelor's degree, 15.9% with master's degree and 7.4% were college graduates. Most hospital dietitians had internship training and 35.2% took a training course of 6 to 12 months. The average space of kitchen was 452.52 $m^2$, with 133.63$m^2$ for modified diet space and 18.13$m^2$ for nutrition counselling room space. The total number of normal meals was 1,255.47, with 502.93 of modified meals. The average calorie of normal meals was 2,145.04kcal, with 91.9g of protein contained in normal meal. The total food waste was 351.40kg. An average monthly cost for disposing food waste was 745,171.67 won. 83.3% of the kitchens were on the 1st basement and only 66.7%(20) of 30 hospitals had its own nutrition counselling room. 80% used dishwashers and 66.7% had an exclusive elevator for delivering meals. 80.0% of hospitals used centralized delivery service. 90% had established an area for hygiene division, 70% used dry zone within the dishwashers for sterilization, 66.7% provided spoon and chopstick, and 100% used dish cover. As means of food waste treatment, most hospitals(56.7%) used animal feed by contracters, followed by means of collection by contracters(30%) and in-house high speed fermentation machines(13.3%). It was found that only 33.3% hospitals regularly checked temperatures of the meals given to patients. Total productivity index was 3.72(meals/hour) in average, with an average productivity index for normal diet of 5.41. Average productivity index for modified diet was 4.62. Productivity indices for patient meals and clinical nutrition were 5.01(meals/hour) and 1.12(cases/hour) respectively and hospitals under self-operated foodservice management received higher points on clinical nutrition productivity index(P<0.01) than hospitals under contract foodservice management.